The Unifying Hypothesis of Alzheimer’s Disease: Heparan Sulfate Proteoglycans/Glycosaminoglycans Are Key as First Hypothesized Over 30 Years Ago

The updated “Unifying Hypothesis of Alzheimer’s disease” (AD) is described that links all the observed neuropathology in AD brain (i.e., plaques, tangles, and cerebrovascular amyloid deposits), as well as inflammation, genetic factors (involving ApoE), “AD-in-a-Dish” studies, beta-amyloid protein (Aβ) as a microbial peptide; and theories that bacteria, gut microflora, gingivitis and viruses all play a role in the cause of AD. The common link is the early accumulation of heparan sulfate proteoglycans (HSPGs) and heparan sulfate glycosaminoglycans (GAGs). HS GAG accumulation and/or decreased HS GAG degradation is postulated to be the key initiating event. HS GAGs and highly sulfated macromolecules induce Aβ 1–40 (but not 1–42) to form spherical congophilic maltese-cross star-like amyloid core deposits identical to those in the AD brain. Heparin/HS also induces tau protein to form paired helical filaments (PHFs). Increased sulfation and/or decreased degradation of HSPGs and HS GAGs that occur due to brain aging leads to the formation of plaques and tangles in AD brain. Knockout of HS genes markedly reduce the accumulation of Aβ fibrils in the brain demonstrating that HS GAGs are key. Bacteria and viruses all use cell surface HS GAGs for entry into cells, including SARS-CoV-2. Bacteria and viruses cause HS GAGs to rapidly increase to cause near-immediate aggregation of Aβ fibrils. “AD-in-a-dish” studies use “Matrigel” as the underlying scaffold that spontaneously causes plaque, and then tangle formation in a dish. Matrigel mostly contains large amounts of perlecan, the same specific HSPG implicated in AD and amyloid disorders. Mucopolysaccharidoses caused by lack of specific HS GAG enzymes lead to massive accumulation of HS in lysosomal compartments in neurons and contribute to cognitive impairment in children. Neurons full of HS demonstrate marked accumulation and fibrillization of Aβ, tau, α-synuclein, and prion protein (PrP) in mucopolysaccharidosis animal models demonstrating that HS GAG accumulation is a precursor to Aβ accumulation in neurons. Brain aging leads to changes in HSPGs, including newly identified splice variants leading to increased HS GAG sulfation in the AD brain. All of these events lead to the new “Unifying Hypothesis of Alzheimer’s disease” that further implicates HSPGs /HS GAGs as key (as first hypothesized by Snow and Wight in 1989).

Tau Protein Interaction Partners and Their Roles in Alzheimer’s Disease and Other Tauopathies

Tau protein plays a critical role in the assembly, stabilization, and modulation of microtubules, which are important for the normal function of neurons and the brain. In diseased conditions, several pathological modifications of tau protein manifest. These changes lead to tau protein aggregation and the formation of paired helical filaments (PHF) and neurofibrillary tangles (NFT), which are common hallmarks of Alzheimer’s disease and other tauopathies. The accumulation of PHFs and NFTs results in impairment of physiological functions, apoptosis, and neuronal loss, which is reflected as cognitive impairment, and in the late stages of the disease, leads to death. The causes of this pathological transformation of tau protein haven’t been fully understood yet. In both physiological and pathological conditions, tau interacts with several proteins which maintain their proper function or can participate in their pathological modifications. Interaction partners of tau protein and associated molecular pathways can either initiate and drive the tau pathology or can act neuroprotective, by reducing pathological tau proteins or inflammation. In this review, we focus on the tau as a multifunctional protein and its known interacting partners active in regulations of different processes and the roles of these proteins in Alzheimer’s disease and tauopathies.

Structurally distinct polymorphs of Tau aggregates revealed by nanoscale infrared spectroscopy

Aggregation of the tau protein plays a central role in several neurodegenerative diseases collectively known as tauopathies, including Alzheimers and Parkinsons disease. Tau misfolds into fibrillar beta sheet structures that constitute the paired helical filaments found in Neurofibrillary tangles. It is known that there can be significant structural heterogeneities in tau aggregates associated with different diseases. However, while structures of mature fibrils have been studied, the structural distributions in early stage tau aggregates is not well understood. In the present study, we use AFM-IR to investigate nanoscale spectra of individual tau fibrils at different stages of aggregation and demonstrate the presence of multiple fibrillar polymorphs that exhibit different secondary structures. We further show that mature fibrils contain significant amounts of antiparallel beta sheets. Our results are the very first application of nanoscale infrared spectroscopy to tau aggregates and underscore the promise of spatially resolved infrared spectroscopy for investigating protein aggregation.

Oral (−)-Epicatechin Inhibits Progressive Tau Pathology in rTg4510 Mice Independent of Direct Actions at GSK3β

Aggregation of the microtubule-associated protein tau into paired helical filaments (PHFs) and neurofibrillary tangles is a defining characteristic of Alzheimer’s Disease. Various plant polyphenols disrupt tau aggregation in vitro but display poor bioavailability and low potency, challenging their therapeutic translation. We previously reported that oral administration of the flavonoid (−)-epicatechin (EC) reduced Amyloid-β (Aβ) plaque pathology in APP/PS1 transgenic mice. Here, we investigated whether EC impacts on tau pathology, independent of actions on Aβ, using rTg4510 mice expressing P301L mutant tau. 4 and 6.5 months old rTg4510 mice received EC (∼18 mg/day) or vehicle (ethanol) via drinking water for 21 days and the levels of total and phosphorylated tau were assessed. At 4 months, tau appeared as two bands of ∼55 kDa, phosphorylated at Ser262 and Ser396 and was unaffected by exposure to EC. At 6.5 months an additional higher molecular weight form of tau was detected at ∼64 kDa which was phosphorylated at Ser262, Ser396 and additionally at the AT8 sites, indicative of the presence of PHFs. EC consumption reduced the levels of the ∼64 kDa tau species and inhibited phosphorylation at Ser262 and AT8 phosphoepitopes. Regulation of the key tau kinase glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9 was not altered by exposure to EC in mice or primary neurons. Furthermore, EC did not significantly inhibit GSK3β activity at physiologically-relevant concentrations in a cell free assay. Therefore, a 21-day intervention with EC inhibits or reverses the development of tau pathology in rTg4510 mice independently of direct inhibition of GSK3β.

Structure of Tau filaments in Prion protein amyloidoses

In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann–Sträussler–Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.

Oxidative Stress Conditions Result in Trapping of PHF-Core Tau (297–391) Intermediates

The self-assembly of tau into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer’s disease (AD) pathogenesis. Numerous post-translational modifications enhance or inhibit tau assembly into NFTs. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins, including the formation of dityrosine (DiY) cross-links. Previous studies have revealed that metal-catalysed oxidation (MCO) using Cu2+ and H2O2 leads to the formation of DiY cross-links in two misfolding proteins, Aβ and α-synuclein, associated with AD and Parkinson’s disease respectively. The effect of MCO on tau remains unknown. Here, we examined the effect of MCO and ultra-violet oxidation to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers that fail to bind thioflavin S, lack β-sheet structure and prevents their elongation into filaments. At a higher concentration, Cu2+ (without H2O2) also facilitates the formation of these tau oligomers. The DiY cross-linked tau oligomers do not cause cell death. Our findings suggest that DiY cross-linking of pre-assembled tau promotes the formation of soluble tau oligomers that show no acute impact on cell viability.

Molecular Processing of Tau Protein in Progressive Supranuclear Palsy: Neuronal and Glial Degeneration

Background: Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are examples of neurodegenerative diseases, characterized by abnormal tau inclusions, that are called tauopathies. AD is characterized by highly insoluble paired helical filaments (PHFs) composed of tau with abnormal post-translational modifications. PSP is a neurodegenerative disease with pathological and clinical heterogeneity. There are six tau isoforms expressed in the adult human brain, with repeated microtubule-binding domains of three (3R) or four (4R) repeats. In AD, the 4R:3R ratio is 1:1. In PSP, the 4R isoform predominates. The lesions in PSP brains contain phosphorylated tau aggregates in both neurons and glial cells. Objective: Our objective was to evaluate and compare the processing of pathological tau in PSP and AD. Methods: Double and triple immunofluorescent labeling with antibodies to specific post-translational tau modifications (phosphorylation, truncation, and conformational changes) and thiazin red (TR) staining were carried out and analyzed by confocal microscopy. Results: Our results showed that PSP was characterized by phosphorylated tau in neurofibrillary tangles (NFTs) and glial cells. Tau truncated at either Glu391 or Asp421 was not observed. Extracellular NFTs (eNFTs) and glial cells in PSP exhibited a strong affinity for TR in the absence of intact or phosphorylated tau. Conclusion: Phosphorylated tau was as abundant in PSP as in AD. The development of eNFTs from both glial cells and neuronal bodies suggests that truncated tau species, different from those observed in AD, could be present in PSP. Additional studies on truncated tau within PSP lesions could improve our understanding of the pathological processing of tau and help identify a discriminatory biomarker for AD and PSP.

A failure of β-amyloid physiological function due to genetic deletion of α7 nicotinic acetylcholine receptors induces an Alzheimer's disease-like pathology

The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer's disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ levels that, in turn, triggers an AD-like pathology. To validate this hypothesis, we studied the age-dependent phenotype of α7 knock out mice. We found that α7nAChR deletion caused an impairment of hippocampal synaptic plasticity and memory at 12 months of age, paralleled by an increase of Amyloid Precursor Protein expression and Aβ levels. This was accompanied by other classical AD features such as a hyperphosphorylation of tau at residues Ser 199, Ser 396, Thr 205, a decrease of GSK-3β at Ser 9, the presence of paired helical filaments and neurofibrillary tangles, neuronal loss and astrocytosis. Our findings suggest that α7nAChR malfunction might precede Aβ and tau pathology, offering a different perspective to interpret the failure of anti-Aβ therapies against AD and to find novel therapeutical approaches aimed at restoring α7nAChRs-mediated Aβ function at the synapse.

Tau protein and its role in Alzheimer’s disease physiopathology: a literature review

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a double proteinopathy: deposition of amyloid-β into plaques and hyperphosphorylation of Tau protein. Objectives: To understand the genetic and molecular aspects of Tau protein and its relationship with Alzheimer’s disease. Methods: We conducted a systematic literature search using Pubmed/ MEDLINE and ClinicalKey databases, applying the descriptors: “Alzheimer Disease” AND “Tau proteins’’ AND Tauopathies, during July and August of 2020. The inclusion criteria were English and Portuguese articles published between 2015 and 2020, with human limited study and free full text, excluding images, books, clinical tests, and narrative reviews. After analyzing titles and abstracts, we selected 12 articles and included 7 additional studies. Results: Mapt, the encoder gene of Tau, is located in the 17q21.3 locus and presents 16 exons that, when transcripted, originates 12 copies of mRNA by alternative splicing and 6 Tau’s isoforms. Tau is a microtubule-associated protein (MAP) responsible for cellular cytoskeleton stabilization and maintenance, promoting neuronal axonal transport. A kinase-phosphatase imbalance turns Tau hyperphosphorylated, disassociating it from tubulin and grouping it into insoluble paired helical filaments, which originates neurofibrillary tangles. The tauopathy’s progress causes neurotransmitter destabilization and neuronal death, inducing AD symptomatic manifestations. Conclusions: Due to the gradual worsening of the disease to more debilitating stages, studies focused on deepening the knowledge of genetic and molecular aspects of Tau protein are viable and promising alternatives to improve the quality of patient’s lives.